The characterization and safety assessment of drugs, drug candidates, environmental chemicals, etc. requires extensive resources, particularly when animal studies are employed. Thus, there is a need to develop new in vitro techniques to predict which compounds pose an increased threat to human health and should therefore be prioritized for further screening and evaluation. Because of the critical role self-renewing stem cells play in tissue function and homeostasis, retention of healthy adult stem cells is crucial for human health. Nevertheless, the different responses of human stem cells compared with terminally differentiated cell types (e.g., primary and transformed cell lines) against drugs, drug candidates, and chemicals have been only sparsely studied. Such information would be vital to identify the toxic effects of such chemicals on the human body and in specific organs. We hypothesize that stem cells may behave in a fundamentally different way from the differentiated cells often used for toxicity studies, and we will address this hypothesis through the use of appropriate model systems. An outcome of this study, then, is that toxicity studies must be expanded to include stem cells, or drugs will proceed into trials with missing and even misleading toxicity information. To test this hypothesis, we propose to develop a high-throughput, microscale 3D cell-based screening tool that will enable rapid and highly quantitative information to be obtained on the effects of drugs, drug candidates, and chemical toxicants on human stem cells; specifically neural stem cells (NSCs) in comparison to adult primary and transformed neural cells, and human mesenchymal stem cells (MSCs). The specific aims of the proposed work are to: 1. Establish a robust 3D cell culture chip platform to grow and differentiate NSCs and MSCs; 2. Perform combinatorial in vitro extracellular matrix development; 3. Develop high-throughput on-chip assays of key signaling pathways in NSCs and MSCs that are critical in proliferation (self-renewal) and differentiation; 4. Validate the 3D cell culture microarray platform for cytotoxicity of stem cells in comparison to terminally differentiated primary cells and transformed cell lines using a series of reference compounds. This information is essential for the development of high throughput predictive toxicology screens for drug discovery and the prioritization of environmental chemicals based on their potential human toxicity.